Method for determination of recognition specificity of virus for receptor sugar chain

This application is a continuation of application Ser. No. 12/065,469 filed Mar. 11, 2009, which is a continuation of PCT/JP2006/316928 filed Aug. 29, 2006, which claims priority of Japanese Applications Nos. 2005-255730 filed Sep. 2, 2005 and 2006-050943 filed Feb. 27, 2006. The entire content of each prior application is expressly incorporated herein by reference.

The present invention is related to a method for determining the recognition specificity of a virus for a receptor sugar chain, a new polymer with sialo-oligosaccharide and a support which can be used in the method, and an effective manufacturing method thereof.

There are numerous symptoms of the influenza, from light symptoms like a common cold to severe (life threatening) symptoms like the Spanish flu. In addition, the influenza is a zoonotic disease, therefore, the avian influenza is recently becoming a major problem. It is known that the range of hosts of influenza viruses extends to many animals species. For example, wild waterfowl such as ducks, domestic fowl such as turkeys, chickens, and quails, pigs, horses, cows, ferrets, whales and seals as well as humans can all become hosts for viruses A.

The coat of the influenza virus is covered with projections of two types of enzyme proteins one of which is HA (hemagglutinins) and the other of which is NA (neuraminidase). HA is a hemagglutininating antigen and at the time of attachment, and invasion to a host cell, it binds with a receptor sugar chain containing sialic acid on the surface of the cell and plays an important role when a viral particle is ingested within the cell.

The antigenecity of an influenza virus is decided by a combination of HA and NA and is divided broadly into three types A, B and C. There are four further subtypes such as the Hong Kong strain which are known among the A type. Conventionally, it is known that a different subtype appears in cycles of about ten years and even within the same subtype, the antigenecity changes little by little every year (antigen shift) in the A type. As a result, it is difficult to produce a vaccine which is completely suitable for an antigenic form and its prevention effects have become problematic.

Meanwhile, among classification of the types of influenza virus, other than the above stated category according to antigenicity, there is also a category according to the differences of binding ability of the influenza virus to the receptor sugar chain (non patent document 1). This category is based on the differences of the mode of binding to sialic acid at an end of a receptor sugar chain and also on the differences in the degrees of recognition, binding ability or affinity of the influenza virus to a receptor sugar chain.

For example, the highly pathogenic avain influenza viruses (such as the H5N1 subtype and H9N1, H7N7) strongly recognize the binding mode of [SAα2-3Galβ-(SA: sialic acid)], but the recognition, biding ability or affinity is low for the binding mode of [SAα2-6Galβ-]. On the other hand, the human influenza A virus and human influenza B virus strongly recognize the binding mode of [SAα2-6Galβ-] but the recognition, biding ability or affinity is low for the binding mode of [SAα2-3Galβ-].

The most effective method for judging the ability of an avian influenza virus to infect humans is the method of recognizing the binding ability of the influenza virus to the receptor sugar chain. That is, even in the case where the avian influenza virus has infected a human that does not mean that a change in the host range will be reflected in a mutation of a gene. However, because a variation in the binding ability to a receptor sugar chain is essential for infection, if the recognition specificity of an influenza virus for a receptor sugar chain or its variation can be easily determined, not only can the type of the influenza virus be determined but also a change of a host infected due to a mutation of the virus or the possibility of a large spread can be predicted.

Conventionally, the Resonant Mirror Detection method is used as a method for determining the recognition specificity of a virus for a receptor sugar chain (patent document 1). In this method, a receptor sugar strain for an influenza virus is immobilized within a cuvette of the Resonant Mirror apparatus and an influenza virus sample is made to react with the receptor sugar chain. Then, a change in the resonant angle which occurs by the binding of the receptor sugar chain and the influenza virus is expressed in a binding curve and the response strength is monitored. It is assumed that the recognition specificity of a virus for a receptor sugar chain can be determined by the strength of this response.

Nevertheless, it is difficult to immobilize a receptor sugar chain to a support in this method. That is, a glycoceramide (sialyl (2-3) neolactotetraosylceramide (avian type), sialyl (2-3) lactotetraosylceramide (avian type), sialyl (2-6) neolactotetraosylceramide (human type) and sialyl (2-6) lactotetraosylceramide (human type) etc.) is used as a receptor sugar chain, a glycolipid which does not bind with the influenza virus is further mixed with the glycoceramide and an immobilized receptor sugar chain is prepared by an extremely cumbersome and complicated method in which this glycolipid mixture is immobilized to the bottom surface within the cuvette. Furthermore, it is necessary to use special and large apparatus of the Resonant Mirror apparatus. As a result, although it can be used in large scale research facilities, it is difficult to use in places where patients arise such as airports, poultry farms and stations etc. or in clinical places such as hospitals.

Recently, it is pointed that the avian influenza virus which is highly toxic will be spreading worldwide and the possibility of a pandemic may be occur by mutation of the virus into a virus (new influenza virus) which infects from a human to another human. As a result, the rapid development of a method which can easily determine the recognition specificity of an influenza virus for a receptor sugar chain using inexpensive and simple instruments is being eagerly desired.

Non patent document 1: Sugar chain recognition process in virus infections (Yasuo Suzuki, Biochemistry Volume 76, No. 3, pp. 227-233, 2004))

Patent Document 1: Japanese Laid Open Patent Publication 2001-264333

Patent Document 2: Japanese Laid Open Patent Publication 2003-73397

Patent Document 3: Japanese Laid Open Patent Publication H10-310610

Patent Document 4: Japanese Laid Open Patent Publication 2003-535965

Patent Document 5: Japanese Laid Open Patent Publication H11-503525

Patent Document 6: Japanese Laid Open Patent Publication 2004-115616